Project Summary Kidney cancer is a common and deadly malignancy, with more than 73,000 new cases and 14,000 deaths estimated to occur in 2019 in the United States. Renal cell carcinoma (RCC) is the most prevalent type, accounting for more than 90% of all kidney cancers. Surgical resection is the treatment choice for localized RCC, however, patients with RCC are often deemed unfit for surgery because of multiple comorbidities. Stereotactic body radiotherapy (SBRT), characterized by the delivery of high doses of ionizing radiation in a few fractions, can overcome the radioresistance of RCC and achieve high therapeutic efficiency. Therefore, SBRT is currently considered as an alternative treatment option to manage non-operable patients. Post-treatment surveillance, established on the belief that detecting asymptomatic disease recurrence offers an optimal opportunity for beneficial intervention, is an integral part of RCC care. However, detecting RCC biomarkers by conventional methods requires large sample volumes and tedious work. No molecular biomarkers have been reliably validated or integrated into daily clinical practice to rationally guide kidney cancer therapy after SBRT. Thus, identifying potential RCC post-treatment biomarkers after irradiation with minimum sample preparation and high sensitivity would be invaluable. Recently, ?liquid biopsy? relying on detecting circulating tumor cells (CTCs), circulating transcripts, RNAs (miRNAs/mRNAs), and tumor-related genes has been investigated as a potential diagnostic and prognostic biomarker for various cancers. Extracellular vesicles (EVs) such as exosomes are difficult to detect and all current approaches to detect EVs have proven to be tedious, expensive, and time consuming. The primary objective of this R21 proposal is to develop and evaluate a newly developed immune-tethered lipoplex nanoparticle (ILN) technology system for preclinical molecular diagnosis. ILNs are designed to provide a simple, non-invasive, and low-cost method for RCC surveillance after SBRT. To achieve this goal, we have developed a biochip with nanoparticles containing specific molecular beacons (MBs) inside and RCC-specific antibody on the surface. The antibodies can capture EVs with specific RCC surface receptors, and then MBs can bind to the target EV RNAs to produce fluorescence that can be recorded by total internal reflection fluorescence microscope (TIRF). This new biochip allows capturing homogeneous EV subgroups secreted from RCC tumor cells, and measuring both EV RNA and membrane protein targets in a single step. If successfully implemented, this diagnostic and surveillance tool, could be highly transformative for the surveillance of patients with cancer.